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Dual Location of the Mitochondrial Preprotein Transporters
B14.7 and Tim23-2 in Complex I and the TIM17:23
Complex in Arabidopsis Links Mitochondrial Activity and
Biogenesis
C W OA
Yan Wang,a Chris Carrie,a Estelle Giraud,a Dina Elhafez,a Reena Narsai,a,b Owen Duncan,a James Whelan,a and
Monika W. Murchaa,1
a Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009, Western
Australia, Australia
b Centre for Computational Systems Biology, University of Western Australia, Crawley 6009, Western Australia, Australia
Interactions between the respiratory chain and protein import complexes have been previously reported in Saccharomyces
cerevisiae, but the biological significance of such interactions remains unknown. Characterization of two mitochondrial
preprotein and amino acid transport proteins from Arabidopsis thaliana, NADH dehydrogenase B14.7 like (B14.7 [encoded by
At2g42210]) and Translocase of the inner membrane subunit 23-2 (Tim23-2 [encoded by At1g72750]), revealed both proteins
are present in respiratory chain complex I and the Translocase of the Inner Membrane 17:23. Whereas depletion of B14.7 by
T-DNA insertion is lethal, Tim23-2 can be depleted without lethality. Subtle overexpression of Tim23-2 results in a severe
delayed growth phenotype and revealed an unexpected, inverse correlation between the abundance of Tim23-2 and the
abundance of respiratory complex I. This newly discovered relationship between protein import and respiratory function was
confirmed through the investigation of independent complex I knockout mutants, which were found to have correspondingly
increased levels of Tim23-2. This increase in Tim23-2 was also associated with delayed growth phenotypes, increased abundance
of other import components, and an increased capacity for mitochondrial protein import. Analysis of the Tim23-2–overexpressing
plants through global quantitation of transcript abundance and in-organelle protein synthesis assays revealed widespread
alterations in transcript abundance of genes encoding mitochondrial proteins and altered rates of mitochondrial protein
translation, indicating a pivotal relationship between the machinery of mitochondrial biogenesis and mitochondrial function.
INTRODUCTION
A variety of multisubunit protein complexes are present on the
mitochondrial outer membrane (Translocase of the Outer Membrane [TOM] and Sorting and Assembly Machinery) (Stroud
et al., 2010) and on the inner membrane (Translocase of the
Inner Membrane 17:23 [TIM17:23] and TIM22) (Rehling et al.,
2004; Mokranjac and Neupert, 2010), which act together to import the wide variety of mitochondrial proteins synthesized in the
cytoplasm. Whereas the protein import apparatus is generally
well conserved across phylogenetic groups (Hewitt et al., 2011),
particularly the pore-forming subunits, which display high levels
of orthology (Dolezal et al., 2006; Carrie et al., 2010a), significant
differences have been observed between the outer mitochondrial
membrane protein import receptors of plants compared with yeast
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Monika W. Murcha
([email protected]).
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www.plantcell.org/cgi/doi/10.1105/tpc.112.098731
and mammals (Perry et al., 2006; Lister et al., 2007). Further differences include the location of the mitochondrial processing peptidase (MPP) in the cytochrome bc1 complex (Braun et al., 1992;
Glaser et al., 1994) in plants compared with yeast and mammals.
In the last decade, studies have revealed the higher order
structure of multisubunit protein complexes on the mitochondrial
inner membrane, in particular, the identification of respiratory chain
supercomplexes (Stuart, 2008; Wittig and Schägger, 2009; Dudkina
et al., 2010). Supercomplex structures between complex I and III,
complex III and IV, dimeric ATP synthase, and even higher supercomplex structures have been described (Stuart, 2008; Dudkina
et al., 2010; Sunderhaus et al., 2010). The functional significance of
these respiratory supercomplexes is not yet clear; however, it has
been proposed that these supercomplexes may enhance electron
flow via substrate channeling, prevent the production of excess
reactive oxygen species, or play a role in maintaining the stability of
respiratory chain complexes (Dudkina et al., 2010; Sunderhaus
et al., 2010). In the case of the ATP synthase complex, the
arrangement of such supercomplexes is proposed to stabilize the
cristae structure that is characteristic of the mitochondrial inner
membrane (Wittig and Schägger, 2009; Dudkina et al., 2010).
Similarly, protein import complexes have also been reported
to form supercomplexes. Namely, the TIM17:23 complex has
been reported to be associated with both the TOM complex and
the presequence-assisted motor complex (PAM) (Schmidt et al.,
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Figure 1. Overview of the PRAT Proteins in Arabidopsis.
(A) Diagrammatic representation of the 10 PRAT proteins previously shown to have a mitochondrial localization. The complex that these proteins have
been identified in is indicated according to previous studies (Meyer et al., 2008; Klodmann et al., 2010, 2011). aa, amino acids.
(B) Comparison of sequences of Tim23-2 and B14.7 from Arabidopsis and Tim23 from Saccharomyces cerevisiae (Sc). The predicted transmembrane
domains are highlighted, and the PRAT domain (G/A)X2(F/Y)X10RX3DX6(G/A/S)GX3G) is indicated. Percentage identity (in gray) and similarity between
Tim23-2, B14.7, and ScTim23.
Figure 2. Interaction between TIM17:23 and Complex I.
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2010). The PAM complex is located on the matrix side of the inner
membrane and pulls precursor proteins through the TIM17:23
translocase into the matrix or inner membrane. Tim21, Tim23, and
Tim50 play a role in facilitating interactions with the outer membrane TOM complex to form a dynamic TOM-TIM supercomplex
(van der Laan et al., 2006; Chacinska et al., 2010). In fact, two
distinct forms of TIM17:23 have been identified, TIM17:23sort and
TIM17:23motor (Schmidt et al., 2010). TIM17:23sort contains Tim17,
23, 50, and 21 and is involved in the lateral insertion of preproteins
into the inner membrane (Schmidt et al., 2010). TIM17:23motor
translocates proteins into the matrix and does not contain Tim21
but does contain the PAM subunits Tim44, HSP70, Mge1, and
Pam 16, 17, and 18 (Schmidt et al., 2010).
There are also a number of reports of respiratory chain
components and protein import complexes interacting or converging via two types of interactions. First, protein subunits in
a respiratory chain complex have been reported to be involved
in protein import, this was initially reported with the finding that
a subunit of the matrix-located MPP (b-MPP) also functions as
one of the core subunits of the cytochrome bc1 complex in
Neurospora crassa (Schulte et al., 1989). Further studies in
plants revealed a unique evolutionary history for MPP in mitochondria (Braun et al., 1992; Glaser et al., 1994; Eriksson et al.,
1996; Brumme et al., 1998; Glaser and Dessi, 1999), where the
core subunits of plant cytochrome bc1 complexes are bifunctional, acting as both a peptidase and as integral subunits of
the cytochrome bc1 complex (Braun et al., 1992; Eriksson et al.,
1996). More recently, it has been reported that the succinate
dehydrogenase subunit 3 in yeast is also a component of the
TIM22 translocase and has an active role in the import of proteins via the carrier import pathway (Gebert et al., 2011). The
second type of interaction is where the multisubunit protein
complexes interact to form supercomplexes; for example, current evidence indicates that Tim21 mediates the interaction
between the TIM17:23 and complex III in yeast (van der Laan
et al., 2006).
Arabidopsis thaliana contains 17 genes encoding proteins that
belong to the Preprotein and Amino acid Transport family
(PRAT) (Rassow et al., 1999), of which 10 proteins are located in
mitochondria, six are located in plastids, and one protein is dual
targeted to mitochondria and plastids (Murcha et al., 2007). Of
the 10 mitochondrial PRAT proteins, phylogenetic analysis suggested that three genes encode Tim17, three genes encode
Tim23, two identical genes encode Tim22, and three genes
encode proteins of unknown function (Murcha et al., 2007). One
of these genes (At2g42210) encoding a protein of unknown
function was subsequently shown to encode the complex I
subunit B14.7 (Meyer et al., 2008; Klodmann et al., 2010;
Klodmann et al., 2011). Complex I from N. crassa, humans, and
Chlamydomonas reinhardtii have also been shown to contain
a subunit that is similar to the PRAT family proteins, namely,
N. crassa 21.3b (GenBank/P25710), human NDUFA11, and
C. reinhardtii v2.0/C_180167 (GenBank/AAS58499) (Nehls et al.,
1991; Carroll et al., 2002; Murray et al., 2003). In Arabidopsis,
this protein has been identified to be a single peripheral subunit
of complex I in two independent studies, both of which identified
B14.7 in stoichiometric amounts by proteomic analysis (Meyer
et al., 2008; Klodmann et al., 2010).
To investigate the extent and implications of any interactions
between the respiratory chain and protein import complexes on
the inner mitochondrial membrane, the locations and the interactions of various components of the TIM17:23 complex in
Arabidopsis were investigated. The biological implications of
interactions between respiratory chain complexes and the mitochondrial protein import machinery are unclear in plants. While
yeast is an excellent simple eukaryotic model system, it differs
from plants in many aspects with reference to mitochondrial
biology, most notably that it lacks a complex I. In this study, we
describe an interaction between TIM17:23 and respiratory
complex I. Furthermore, the stoichiometric amounts of these
two complexes are linked in vivo and may provide a link between
protein import and respiration to regulate mitochondrial biogenesis in plants.
RESULTS
Interaction between Tim23 and Complex I
Of the 10 mitochondrial PRAT proteins (Murcha et al., 2007), two
have been reported to be located in complex I of the respiratory
chain (Figure 1A). Tim22 was shown to migrate with complex I,
Figure 2. (continued).
(A) Yeast two-hybrid interaction assays indicate a positive interaction of Tim23-2 with complex I subunit B14.7. The yeast strain AH109, transformed
with pGADT7 Tim23-2, was mated with yeast strain Y187, transformed with either pGBKT7 Tim17-2, Tim23-2, Tim50, Tim21, complex III subunit MPPa,
complex I subunit B14.7, or empty vector pGBKT7. Diploid yeast growth on –Trp –Leu media indicates successful mating. Growth on –Trp –Leu –Ade –
His with X-a-D-galactoside indicates specific protein–protein interactions.
(B) Immunodetection of Tim23-2, Tim17-2, B14.7, and Tim50 in total mitochondrial proteins separated by BN-PAGE. Nonspecific bands are indicated
by an asterisk.
(C) BN-PAGE of time-course analysis of the import into mitochondria of radiolabeled Tim23-2, Tim17-2, complex I subunit B14.7, and complex I subunit
encoded by At1g47260. Incorporation of radiolabeled protein into complex I is indicated by arrows, nonspecific incorporation into complex V is
indicated by asterisks, and nonspecific incorporation of B14.7 into an unknown complex is indicated by a plus sign.
(D) Mitochondrial proteins isolated from Col-0 (wild-type) plants were incubated with or without antibodies raised against Tim23-2 and Protein ASepharose. The wash and pellet fractions were resolved by SDS-PAGE and immunodetected with antibodies against Tim23-2, B14.7, complex I subunit
Ndufs4, complex III subunit RISP, and cytochrome c.
(E) Interaction of Tim23-2 with Tim17-2 and B14.7 was determined by BiFC in onion epidermal cells using biolistic transformation. YFP, yellow
fluorescent protein.
Figure 3. Overexpression of Tim23-2 Results in a Retarded Growth Phenotype.
(A) Growth phenotypes for Arabidopsis plants that contain T-DNA insertions annotated to be located in Tim23-2 (At1g72750) at 2 weeks old.
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while the complex I subunit B14.7 (Meyer et al., 2008; Klodmann
et al., 2010), migrates with both complex I and the TIM17:23
complex (Klodmann et al., 2011; Rode et al., 2011). A comparison of B14.7, Tim23-2, and yeast Tim23 shows that B14.7 is
similar to yeast Tim23 in terms of overall characteristics and
contains an Arg residue in the correct position of the PRAT
consensus domain, G/AX2F/YX10RX3DX6G/A/SGX3G, compared
with Tim23-2 (Figure 1B). It has been shown that this Arg residue
must be inserted into Tim23-2 from Arabidopsis before it can
complement a tim23 yeast deletion strain (Murcha et al., 2003).
To confirm that B14.7 was present in both complex I and
TIM17:23, a variety of assays were used both to test for interactions of B14.7 with TIM17:23 and to determine whether B14.7
was a subunit of TIM17:23.
Proteins of the TIM17:23 complex from Arabidopsis were
used to probe for protein–protein interactions using yeast twohybrid assays. Tim23-2 was tested for interactions against
various components of the TIM17:23 complex and complex I
and complex III subunits (Figure 2A). Proteins known to interact
with Tim23-2, such as Tim23-2 itself, Tim17-2, and Tim50,
displayed positive interactions, evidenced by growth on –Leu
–Trp –Ade –His with X-a-D-galactoside and a blue phenotype
(Figure 2A). Tim23-2 was found to interact with the complex III
subunit, MPPa, and the complex I subunit, B14.7 (Figure 2A)
(Meyer et al., 2008; Klodmann et al., 2010). The empty vector
alone exhibited growth only on –Leu –Trp media, confirming that
the growth observed on selection media was due to specific
interactions of Tim23-2 (and Tim17-2) with partner proteins.
Whereas the yeast two-hybrid assays showed that B14.7 can
interact with Tim23-2 (Figure 2A), this does not directly indicate
that B14.7 is a subunit of TIM17:23 or confirm the previous report that B14.7 comigrates with TIM17:23 (Klodmann et al.,
2011; Rode et al., 2011). This was determined in two ways: by
probing protein complexes separated by Blue-Native PAGE
(BN-PAGE) with a variety of antibodies (Figure 2B) and by analyzing the ability of radiolabeled proteins to be incorporated into
protein complexes (Figure 2C). Antibodies raised against Tim23-2
detected two protein bands with apparent molecular masses of
;100 and ;200 kD (Figure 2B). In addition to several nonspecific interactions (indicated by an asterisk), a band was
consistently detected at ;720 kD, which represents the mobility
of monomeric complex I. Probing with Tim17-2 antibody detected the single TIM17:23 complex at ;200 kD, and no crossreaction was observed at ;720 kD, which corresponds with the
location of complex I (Figure 2B). The antibody raised against
B14.7 similarly cross-reacted with the Tim17:23 complex at
;100 and ;200 kD in addition to the monomeric complex I
(Figure 2B). The specificity of the antibody to B14.7 was determined against recombinant full-length Tim23-1, Tim23-2, and
Tim23-3 proteins, and no cross-reactivity was observed (see
Supplemental Figure 1 online). An antibody raised against Tim50
also detected the upper band of Tim17:23 complex, but not
complex I (Figure 2B). To confirm further the dual locations of
both Tim23-2 and B14.7, in vitro protein uptake assays of radiolabeled proteins were performed and analyzed by BN-PAGE.
The uptake of radiolabeled Tim23-2 into the Tim17:23 complexes
was observed as expected, but uptake of Tim23-2 into the
monomeric complex I was also distinctly observed, in a manner
that increased over time (Figure 2C). In the case of Tim17-2,
whereas import into the TIM17:23 complexes was similarly observed, there was no incorporation of radiolabeled protein into
complex I (Figure 2C). For B14.7, uptake was observed into the
TIM17:23 complex and complex I in a time-dependant manner,
whereas another complex I subunit, encoded by At1g47260,
was shown to incorporate into both monomeric complex I and
the supercomplex I+III (Figure 2C). Incorporation of B14.7 was
also observed into an unknown complex, located between
complex III and complex IV (Figure 2C, indicated with a +). In
addition, nonspecific labeling of complex V was observed with
all precursor proteins (indicated by an asterisk), which likely
represents the incorporation of radiolabeled Met into subunit 9
and the a-subunit of the ATP synthase complex that are encoded in the mitochondrion. While unlabeled Met is added prior
to the import assay, it is not unexpected that some incorporation
can still occur (Carrie et al., 2010b). These results suggest that
both Tim23-2 and B14.7 are imported into and accumulate in
both complex I and the TIM17:23 complex.
The interaction of Tim23-2 with complex I was further confirmed by immunoprecipitation and bimolecular fluorescence
complementation (BiFC) (Figures 2D and 2E). Mitochondria
isolated from Columbia-0 (Col-0) plants were lysed and immunoprecipitation was performed with antibodies raised against
Tim23-2. In addition to Tim23-2 and B14.7 being detected in the
precipitated products (Figure 2D), the complex I subunit Ndufs4
and complex III subunit Rieske Iron-Sulfur protein (RISP) were
also detected, while cytochrome c was not, indicating that antibodies to Tim23-2 can specifically pull down complex I and
complex III subunits. Using a split yellow fluorescent protein
BiFC assay, the interaction of Tim23-2 with the B14.7 was
confirmed and found to be mitochondrial in location by biolistic
transformation in onion (Allium cepa) epidermal cells (Figure 2E).
Finally, it has been reported previously that in yeast, Tim21
interacts with complex III (van der Laan et al., 2006). Using
a similar set of assays, interaction between Tim21 (encoded by
At4g00026) and the cytochrome bc1 complex was revealed in
Arabidopsis (see Supplemental Figure 2 online). However, as
yeast mitochondria lack complex I, and instead use a type II NADH
dehydrogenase (Melo et al., 2004), this interaction between
Figure 3. (continued).
(B) The positions of the T-DNA inserts in SALK_143656 and GABI_689C11 lines. UTR, untranslated region.
(C) Mitochondria isolated from wild-type (Col-0), SALK_143656 (tim23-2 OE), and GABI_689C11 (tim23-2 KO) were subjected to SDS-PAGE and
probed with antibodies raised against Tim23-2 and Porin (see Supplemental Figure 3A online). The apparent molecular mass of the protein detected is
indicated. The 20-kD band is consistent with previous analysis of in vitro synthesized and imported Tim23-2 (Murcha et al., 2005b). Coomassie blue
staining shows equal loading of protein.
A Link between Mitochondrial Activity and Biogenesis
complex I and TIM17:23 has not been previously shown in
other systems. Therefore, we further investigated this interaction using genetic approaches to determine the biological significance.
Genetic Characterization of the Interactions between
Tim17-2, Tim23-2, and B14.7
To characterize the interactions between Tim17-2, Tim23-2, and
B14.7, T-DNA insertion lines were screened for each protein. In
the case of Tim17-2 and B14.7, no homozygous lines could be
obtained, indicating these are essential proteins in Arabidopsis.
As tim17 is an essential gene in yeast, it is not unexpected that
such lethality was also observed in Arabidopsis (Dekker et al.,
1993; Maarse et al., 1994). Interestingly, previous studies have
characterized complex I–deficient mutants from tobacco (Nicotiana tabacum) and Arabidopsis and revealed that these plants
are viable but exhibit a retarded growth phenotype (Dutilleul
et al., 2003; Meyer et al., 2009). Therefore, the lethality of the
knockout for B14.7 suggests that it may have other essential
roles, in addition to being a complex I subunit.
Two viable T-DNA insertion lines (SALK_143656 and
GABI_689C11) were obtained for Tim23-2 (Figure 3A). However,
while one line displayed an apparently normal growth phenotype
(GABI_689C11), the other line (SALK_143656) displayed a severe delayed growth phenotype throughout the life cycle (Figure
3A). This apparent discrepancy was resolved by mapping and
sequencing of the T-DNA insertion in both lines, which revealed
that whereas one line (GABI_689C11) represented an insertion in
the gene at position 43 bp 39 (downstream) of the translational
start site, the insertion for the other line (SALK_143656) was in
fact located 135 bp 59 (upstream) of the translational start site
(Figure 3B). To determine the effect of both insertions on the
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abundance of Tim23-2 protein, antibodies raised against Tim23-2
were used in quantitative immunoblot analysis of mitochondria
isolated from each line (Figure 3C). In SALK_143656, in which
the T-DNA was inserted 135 bp upstream of the translation start
site and which exhibited a retarded growth phenotype (Figures
3A and 3B), there was a significant increase (ranging from 1.7- to
2.25-fold; P < 0.05) in abundance of Tim23-2 protein (Figure 3C).
By contrast, the same Tim23-2 protein quantitation in the T-DNA
insertion line GABI_689C11, where the T-DNA insert is located
at a position 43 bp 59 of the gene, resulted in no Tim23-2 protein
detected with an apparent molecular mass of 20 kD; instead,
a smaller unique band of 18 kD was detected (indicated by an
asterisk, Figure 3C). Thus, these lines are referred to as knockout
(tim23-2 KO) for GABI_689C11 and overexpressor (tim23-2 OE)
for SALK_143656.
The specificity of the Tim23-2 antibody, which was raised
against amino acid regions 3 to 32 and 103 to 143, was verified
using recombinant full-length overexpressed protein for all three
Tim23 isoforms (see Supplemental Figure 3A online). The Tim23-2
antibody cross-reacted against both recombinant Tim23-1
and Tim23-2. Thus, it was not possible to distinguish between
Tim23-1 and Tim23-2 using the Tim23-2 antibody alone. An
additional antibody raised against Tim23-3 (amino acids 6 to 32
and 64 to 132) detected both recombinant Tim23-1 and Tim23-3
proteins but not Tim23-2 (see Supplemental Figure 3A online).
Using the Tim23-3 antibody, no signal was detected in immunoblot analysis of mitochondria isolated from Col-0 plants or any
of the Tim23 T-DNA lines (see Supplemental Figure 3A online),
implying that Tim23-1 and Tim23-3 are not present in detectable
amounts in mitochondria under the conditions tested in this
study.
Notably, probing mitochondria isolated from the tim23-2 KO line
with the Tim23-2 antibody detected a weak band, corresponding
Figure 4. Complex I Is Reduced in tim23-2 OE Mutant.
Mitochondria were isolated from Col-0 (wild-type), tim23-2 OE, and tim23-2 KO lines and resolved by one-dimensional BN-PAGE, stained with
Coomassie blue, and analyzed for complex I activity. The bands corresponding to the supercomplex I+III, complex I, complex V, complex III, and
complex IV are indicated. Immunodetection of complex I subunit Ndufs4, complex V a-subunit of ATP synthase, complex III subunit RISP, and complex
IV subunit cytochrome oxidase II (COXII). The presence of an additional complex in the tim23-2 OE line termed MIC was also detected by Coomassie
blue staining and the RISP antibody as indicated with a black box.
Figure 5. Growth Stage Analysis of tim23-2 OE, tim23-2 KO, Complex I Mutant Lines, rug, ndufs4, and rpotmp and tom20 Triple Mutant.
A Link between Mitochondrial Activity and Biogenesis
to a smaller protein of ;18 kD in size, which is not evident in
Col-0 or tim23-2 OE blots (Figure 3C). Analysis of the transcript
abundance for Tim23-2 revealed that for the tim23-2 KO line, the
T-DNA insertion at position 43 bp results in elevated amounts of
Tim23-2 transcript downstream of the T-DNA insert. This is in
agreement with the detection of a smaller protein likely to represent a truncated form of Tim23-2, translated downstream of
the T-DNA insert (see Supplemental Figure 3B online). In the
case of the tim23-2 OE line, while Tim23-2 transcript abundance
was not elevated, and even somewhat reduced (see Supplemental Figure 3B online), protein abundance was increased
(Figure 3C). Previous studies in rice (Oryza sativa) and Arabidopsis have reported that changes in the protein abundances of
components of the mitochondrial protein import apparatus are
generally not accompanied by changes in the respective transcript abundances. This was observed during germination in rice
(Howell et al., 2006) and between aerobically and anaerobically
grown rice (Howell et al., 2007). Similarly, in Arabidopsis, the
mitochondrial protein import components do not show a correlation between changes in protein and transcript abundance
during germination (Law et al., 2012). In addition, the inactivation
of any Tom20 or OM64 genes, which encode the receptor
components of the TOM complex, has been shown to result in
an increase in protein abundance of other isoforms, without any
change seen in transcript abundance (Lister et al., 2007).Thus,
the amount of Tim23-2 in the tim23-2 OE line appears to be
regulated posttranscriptionally.
To investigate further possible reasons for the observed
growth defect of tim23-2 OE plants, mitochondria from Col-0,
tim23-2 OE, and tim23-2 KO lines were analyzed by BN-PAGE.
Analysis of the Coomassie blue–stained gel showed that there
was a difference in the banding pattern observed for Col-0,
tim23-2 OE, and tim23-2 KO mitochondria (Figure 4). Mitochondria from Col-0 plants showed the characteristic pattern of
the respiratory chain complexes, with both the monomeric
complexes I and III and the supercomplex I+III evidenced (Figure
4). The monomeric form of complex I appears to be absent in the
tim23-2 OE line, while the supercomplex I+III is greatly reduced
in abundance (Figure 4). An additional band, between the supercomplex I+III and complex I was observed (Figure 4, black
square) and termed mixed intermediate complex (MIC), which
comprises a mixture of inner membrane complex components,
such as subunits of complex I, III, V, and prohibitin (see Supplemental Figure 4 online). The pattern of protein complexes
from tim23-2 KO plants were similar to Col-0 plants, except that
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the monomeric form of complex I appeared slightly diffused
(Figure 4). An activity stain for complex I, analyzed by BN-PAGE,
confirmed the reduction of complex I in tim23-2 OE, although it
is notable that some activity is still present, especially at the
supercomplex I+III (Figure 4). Immunoblot analysis, using an
antibody specific for the complex I subunit, Ndufs4, confirmed
that complex I was severely reduced in the tim23-2 OE line
(Figure 4). The mobility and amount of complex IV was unchanged when detected with antibodies against the complex IV
subunit, COXII. Probing with antibodies against the complex III
subunit, RISP, showed a reduction in the supercomplex of
complex I+III and the presence of this subunit in the MIC complex (Figure 4). Also, while the intensity of complex III appears to
have increased, as determined by the intensity of the band
probed with the RISP antibody, this may be explained in part
by the absence of the supercomplex I+III. An increase in the
abundance of the complex V a-subunit of ATP synthase was
also observed in the tim23-2 OE line (Figure 4).
The data above suggests that the growth defect observed
within the tim23-2 OE plants occurred as a result of a reduction
in the amount of complex I. Thus, we characterized several other
mutant lines where complex I activity had been dramatically
reduced. Three independent complex I mutants, rug, ndufs4,
and rpotmp, were used and growth analysis was performed and
compared with tim23-2 OE plants. Additionally, as tim23-2 KO
appeared to have no deleterious growth defect, it was also included in the growth analysis. Finally, a triple mutant for the
tom20 outer membrane preprotein receptor that displays significantly reduced rates of protein import (Lister et al., 2007) was
also used for comparison. The tim23-2 OE line displayed
a similar delayed growth phenotype to the complex I mutants
when grown on plates and soil (Figures 5A and 5B), and as
expected, the tim23-2 KO plants displayed growth characteristics more similar to Col-0 (Figures 5A and 5B), reaching defined development stages earlier than Col-0 (Figure 5B), with
plant height, and maximum rosette radius even observed to be
significantly greater than Col-0 (P < 0.05) (Figure 5C). Based on
these findings, phenotypically the tim23-2 OE is most similar to
complex I mutants.
Overexpression of Tim23-2 and Complex I Mutants Shows
Increased Rates of Protein Import
As Tim23 is the channel forming subunit in the TIM17:23 translocase in yeast (Truscott et al., 2001), we investigated whether
Figure 5. (continued).
(A) Plate-based growth progression analysis of Col-0. tim23-2 KO, tom20-2 20-3 20-4 (tom20 triple mutant), tim23-2 OE, rug ndufs4, and rpotmp.
Arrows define the time (days after sowing) that Col-0 plants have reached the growth stages as defined by Boyes et al. (2001). Boxes define the time
between the growth stages, and shading indicates the occurrence of each growth stage. Stage 0.1, imbibition; stage 0.5, radical emergence; stage 0.7,
hypocotyl emerge from seed coat; stage 1.0, cotyledons fully opened; stage 1.02, two rosette leaves >1 mm in length; stage 1.04, four rosette leaves >1
mm in length. Data are given as averages for 10 plants. Days are relative to the days after sowing after a 3-d stratification at 4°C
(B) Soil-based growth progression as in (A): stage 1.10, 10 rosette leaves >1 mm; stage 5.10, first flower buds visible; stage 6.00, first flower opens;
stage 6.90, flowering complete.
(C) Representative growth parameters of Col-0 and mutants, number of rosette leaves >1 mm, maximum rosette radius (mm) over time, plant height
(cm) over time, and root length (cm) at 14 d. Data are given as averages 6 SE for 10 plants. P < 0.05 using a Student’s t test.
Figure 6. Overexpression of Tim23-2 or a Decrease in Complex I Results in Increased Import Ability.
A Link between Mitochondrial Activity and Biogenesis
protein import was affected in the tim23-2 KO and OE lines in
Arabidopsis. Three proteins that contain N-terminal cleavable
targeting signals and are imported via the general import pathway were used including the alternative oxidase (AOX) (Whelan
et al., 1995), the FAd subunit of ATP synthase (FAd) (Smith et al.,
1994), and the dual-targeted protein monodehydroascorbate
reductase (MDHAR) (Chew et al., 2003). Protein import via the
carrier import pathway was also tested using the adenine nucleotide carrier (ANT) and the phosphate carrier (Pic) (Murcha
et al., 2005b). The tim23-2 OE line exhibited an increase in the
import ability of AOX, FAd, and MDHAR, while mitochondria
isolated from tim23-2 KO plants displayed a reduced import
ability of these three precursor proteins (Figure 6A). The effect of
altered Tim23-2 levels was seen to be specific to precursor
proteins imported via the general import pathway, as no differences were observed with the import of ANT or Pic (Figure 6A),
which are both precursors of the carrier import pathway (Murcha
et al., 2005b). Thus, while overexpression of Tim23-2 protein
resulted in an increased import ability via the general import
pathway, it is not clear why a retarded growth phenotype was
observed.
Characterization of the import capacity of mitochondria isolated from the complex I mutants also displayed an increase in
protein import (Figures 6B and 6C). The rug mutant line revealed
increased import ability of all proteins tested via both the general
import pathway (AOX, FAd, and MDHAR) and the carrier import
pathway (ANT and Pic) (Figure 6B). The latter differs from the
tim23-2 OE line, in that there was no increase in the import ability
of carrier pathway proteins (Figure 6A, ANT and Pic panels versus
Figure 6B, ANT and Pic panels). Similarly, the import ability of the
ndufs4 mutant line and to a lesser extent the rpotmp line, exhibited increased import ability of AOX, MDHAR, and Pic precursor proteins when compared with Col-0 (Figure 6C).
A Reduction in Complex I Results in an Increase in Tim23
To further investigate the similarities between the tim23-2 OE
plants and the complex I mutant plants, mitochondria were
isolated from Col-0, tim23-2 KO, tim23-2 OE, rug, ndufs4, and
rpotmp plants and probed with a series of antibodies raised
against mitochondrial PRAT proteins (Murcha et al., 2007), respiratory chain components, and proteins involved in protein
import (Figure 7). Antibody dilutions were optimized for specific
concentrations to ensure linearity of detection against Col-0
mitochondria (see Supplemental Figure 5 online). Interestingly,
11 of 21
probing mitochondria isolated from these lines, with antibodies
to Tim23-2, revealed that all complex I mutants displayed an
increase in Tim23-2 protein abundance, similar to that of mitochondria isolated from the tim23-2 OE plants (Figure 7A).
In the case of other mitochondrial PRAT proteins, it was evident that an increase or decrease in the amount of Tim23-2 was
accompanied by similar variations in Tim17-2 protein abundance (Figure 7A). For B14.7, a subunit of complex I and a PRAT
protein, abundance decreased only slightly in the tim23-2 KO
line and increased in the tim23-2 OE and ndufs4 lines. B14.7
protein was not detected in the rug mutant plants and was only
barely detected in the rpotmp mutant plants (Figure 7A). This
indicates that while a T-DNA insertion into the locus At2g42210
(encoding B14.7) is lethal, even the reduced amounts of the
protein that were detected in the rug line and rpotmp lines are
sufficient to restore viability. As complex I is still largely absent in
these lines, it further supports a dual role for B14.7, one in
complex I and a second role in the TIM17:23 complex, that is
essential for viability. A PRAT protein of unknown function encoded by At3g25120 showed slight increases in abundances in
all lines, including in the tim23-2 KO line (Figure 7A). Tim22,
a protein encoded by At3g10110 or At1g18320 (two genes
which have 100% identity), which has been shown to complement a yeast tim22 deletion mutant (Murcha et al., 2007), also
increased in abundance in the tim23-2 OE and complex I mutant
lines (Figure 7A).
Analysis of a variety of protein import components revealed
an increase in Tim44 protein abundance in all lines (Figure 7B). In
addition, all three Tom20 isoforms increased in abundance in all
lines. Increases were also observed in the abundance of Sam50,
Tim50, Tim21, and Tim9, most prominently in the tim23-2 KO,
OE, and rug lines (Figure 7B).
In the case of a variety of other respiratory chain components,
the most notable effect observed was a >80% reduction in
Ndufs4 protein abundance in the tim23-2 OE, rug, and rpotmp
lines (Figure 7C), supporting the previous report of this for mitochondria from rug lines (Kühn et al., 2011). Ndufs4 is a complex I subunit that is proposed to be present in the peripheral
portion of complex I (Klodmann et al., 2010; Klodmann and
Braun, 2011). Notably, Nad9, a mitochondrial encoded complex
I component, present in the matrix arm, Q module (Klodmann
et al., 2010; Klodmann and Braun, 2011) was also slightly decreased in abundance, in the complex I mutants, though it was
surprisingly higher in abundance in the tim23-2 OE line (Figure
7C). A variety of other respiratory chain components increased
Figure 6. (continued).
(A) In vitro uptake assays into mitochondria isolated from Col-0, tim23-2 OE, and tim23-2 KO lines. Import of [35S]Met-radiolabeled AOX, FAd subunit of
ATP synthase (FAd), MDHAR, ANT, and Pic into isolated mitochondria. Aliquots where removed at 5-, 10-, 15-, and 20-min time points and treated with
Proteinase K. The precursor (p) and mature (m) form are indicated.
(B) As in (A) except mitochondria were isolated from the rug mutant.
(C) As in (A) except mitochondria were isolated from complex I mutants ndufs4 and rpotmp lines and imports performed using AOX, MDHAR, and Pic as
described above except that 3-, 6-, and 12-min time points were used. All import assays were carried using 50 mg of isolated mitochondria as
determined by protein assay prior to import. The rate of import of the Proteinase K–protected proteins was determined at all time points and normalized
to Col-0 (wild-type) at the highest time point for each replicate experiment (n $ 3 6 SE).
[See online article for color version of this figure.]
Figure 7. The Abundance of a Variety of Proteins as Determined by Immunoblot Analysis of Mitochondria Isolated from Col-0 (Wild Type), tim23-2 OE,
tim23-2 KO, and rug, ndufs4, and rpotmp Lines.
A Link between Mitochondrial Activity and Biogenesis
in abundance to some extent across the tim23-2 KO, tim23-2
OE, and complex I mutant lines, while the a- and b-subunits of
the ATP synthase complex increased in abundance most
prominently in the tim23-2 OE, rug, and ndufs4 lines (Figure 7C).
COXII protein levels similarly increased in these lines, with barely
detectable amounts observed in rpotmp (Figure 7C). The Rieske
FeS protein of the cytochrome bc1 complex showed a similar
increase in the tim23-2 OE, rug, and ndufs4 lines (with no increase in the rpotmp line) (Figure 7C). Equal loading of total
mitochondrial proteins was confirmed with Coomassie blue
staining of mitochondrial samples (see Supplemental Figure 5B
online), and no changes were observed in the abundance of
porin and TOM40 (Figures 7B and 7C). t tests and calculation of
coefficients of variation (CV) showed no significant (P < 0.05)
changes and very low variability in protein abundance for both
porin (CV < 0.1) and TOM40 (CV < 0.1), confirming equal total
mitochondrial protein loading.
Analysis of Global Transcriptional Changes in Tim23-2
Mutant Plants
tim23-2 OE lines displayed extensive transcriptional reprogramming, with a total of 1645 transcripts significantly altered
with respect to Col-0 seedlings (861 transcripts upregulated and
784 transcripts downregulated >1.5-fold, after false discovery
rate [FDR] correction [P < 0.05, posterior probability of differential expression (PPDE) (<P) > 0.95]) (Figure 8A). tim23-2 KO
lines displayed fewer significant changes, with 525 and 466
transcripts significantly down- and upregulated >1.5-fold, respectively, after FDR correction. Comparison of transcript responses between the tim23-2 OE and the complex I mutant rug
lines showed significant overlap, not only in the specific genes
altered, but also the direction and magnitude of the responses.
Significantly more transcripts were coexpressed between rug
and tim23-2 OE than would be expected by random chance,
(P < 0.001, x2 test) (indicated by #, Figure 8A). This indicates that
the transcriptional responses of rug and tim23-2 OE show
significant similarity on a genome-wide scale. Transcript
abundance changes for components specifically related to
mitochondrial import functions were examined in the two
mutant lines. For both lines, transcripts encoding mitochondrial intermembrane space chaperones and factors associated with folding in the matrix were consistently upregulated
(see Supplemental Table 1 online; Figure 8B).
To investigate further the transcriptomic changes in the tim23-2
OE mutants in relation to mitochondrial function and regulation,
significant differential expression was visualized on a custom
13 of 21
MapMan image, displaying diverse facets of mitochondrial
function and biogenesis (Figure 8B). Overexpression of Tim23-2,
and the associated complex I defect, appears to induce a substantial response within the mitochondrion, including the upregulation of numerous nucleus-encoded and organelle-encoded
components of all five cytochrome respiratory pathway complexes (Figure 8B). This is accompanied by an upregulation,
at the transcript level, of nuclear-encoded mitochondrial transcription/translation machinery, mitochondrial import components, and a specific upregulation of reactive oxygen species/
REDOX scavenging systems within the mitochondria, most likely
due to an increase in potential for reactive oxygen species production, due to respiratory chain dysfunction (Figure 7C). Furthermore, steady state mitochondrial encoded transcript levels
were measured by quantitative RT-PCR, and a fourfold to
10-fold increase was observed for almost all transcripts encoded by the mitochondrial genome (Figures 8B and 8C) This
increase in mitochondrial transcription was also accompanied
by an increase in the rates of mitochondrial translation, evident
for a number of mitochondrial encoded proteins (Figure 8D).
Together, these results suggest that overexpression of Tim23-2
protein results in the instability of mitochondrial respiratory chain
complex I and signals a mitochondrial biogenesis response to
induce transcription/translation of all components necessary to
build a functional organelle.
This potential regulation of Tim23-2 with mitochondrial biogenesis is further supported by genome-wide analysis of the
coexpression environment of Tim23-2. The top 50 coexpressed
Arabidopsis genes with Tim23-2 (based on r values across >300
public microarray data sets) from the BAR expression Angler
and ACT (http://www.Arabidopsis.leeds.ac.uk/act/coexpanalyser.
php) were combined, leaving a list of 76 unique genes (see
Supplemental Table 2 online). Figure 8D shows a functional categorization of this list, compared with the whole genome, and
genes are significantly (P < 0.0001) overrepresented in the functional categories of protein synthesis (15.8% versus 2.6%), development (11.8% versus 2.8%), protein targeting (6.6% versus
1.1%), RNA binding (6.6% versus 0.7%), RNA processing (6.6%
versus 1.1%), cell division (2.6% versus 0.4%), and protein
folding (2.6% versus 0.3%), supporting a potential role for Tim23-2
during mitochondrial biogenesis.
DISCUSSION
The results presented indicate that there is an interaction between
complex I of the respiratory chain and TIM17:23 in Arabidopsis. It
Figure 7. (continued).
(A) PRAT family members: Immunodetection with antibodies raised against various proteins that belong to the mitochondrial PRAT family. Note that
B14.7 is classified as being a member of the PRAT family (Rassow et al., 1999) and a subunit of complex I (Meyer et al., 2008; Klodmann et al., 2010).
The protein encoded by At3g25120 has a yet unknown function.
(B) Immunodetection with antibodies to known mitochondrial protein import components.
(C) Immunodetection assays were also completed with antibodies raised against various respiratory chain components representing each complex.
Porin was used as a loading control for total protein abundance. Graphs to the right represent the average relative abundance ratios of proteins present
in each mutant compared with Col-0 at 20 mg (blue bar graph) and 40 mg (red bar graph) of total mitochondrial protein (n $ 3 6 SE). Standard errors for
these average ratios are indicated on each graph.
Figure 8. Global Analysis of Transcript Abundance Changes in tim23-2 OE and KO Lines with Other Mutants Affecting Complex I.
A Link between Mitochondrial Activity and Biogenesis
appears that Tim23-2 and B14.7 proteins are both imported into
and accumulate in the respiratory complex I and the TIM17:23
complex. Whereas previous studies in yeast have shown interactions between the respiratory chain and protein import complexes, this study reveals an interaction between the protein
import components and complex I. It is likely that this interaction
was not previously detected simply due to the fact that yeast
lack complex I (Melo et al., 2004). In an attempt to investigate
further the biological significance of Tim23-2 and B14.7 being
present in both complexes, T-DNA insertion lines were screened
for B14.7 and Tim23-2. Two homozygous T-DNA insertion lines
for Tim23-2 were obtained, and despite repeated self-pollination
of heterozygous plants, no homozygous lines for B14.7 could be
obtained. For the Tim23-2 T-DNA insertion lines, one line produced TIM23-2 OE plants, while the other line produced authentic KO plants.
None of the other Tim23 isoforms (i.e., Tim23-1 or Tim23-3)
were detected in the tim23-2 KO plants, suggesting that the
reason this T-DNA KO was not lethal was because B14.7 could
at least partially fulfill the role of Tim23-2. Note that although the
tim23-2 KO plants were viable, a reduced rate of protein import
via the general import pathway was seen, indicating that B14.7
could not completely complement the function of Tim23-2.
However, a reduced rate of import appears to have no deleterious effect on the growth phenotype; in fact, tim23-2 KO plants
even grew slightly better than Col-0 (Figure 5). It is notable that
while Tim23-2 and B14.7 proteins do not display high levels of
sequence identity (20 and 33%, respectively), this is not an indication that complementation cannot occur. For example, while
Tim23 and Tim17 from yeast and plants do not display high
levels of sequence identity, the plant genes for these components can still complement yeast deletion strains and restore
viability (Murcha et al., 2007), and B14.7 does have a consensus
PRAT domain, whereas Tim23-2 does not (Rassow et al., 1999).
Finally even low levels of B14.7 are sufficient to maintain viability, as observed in the rug and rpotmp KO lines, where only
low levels of B14.7 are observed (Figure 7A) (Kühn et al., 2009,
2011). The amount of the TIM23 complex is quite low compared
with respiratory chain complexes, even in yeast, where genetic
approaches had to be used to characterize TIM translocases,
15 of 21
compared with the more abundant TOM components, which
could be characterized by biochemical approaches (Rehling
et al., 2001).
Given the nature of this tim23-2 OE line in Arabidopsis, it was
not possible to complement this mutant, and previous attempts
to overexpress Tim23-2 using the 35S cauliflower mosaic virus
promoter have proved unsuccessful. Therefore, to verify the
findings that an increase in Tim23-2 protein abundance resulted
in a reduction of complex I, we investigated a number of additional mutants that also exhibit a reduction in complex I activity,
including the complex I rug mutant, a nad2 splicing mutant
(Kühn et al., 2011), the rpotmp mutant (Kühn et al., 2009), and
the ndufs4 mutant (Meyer et al., 2009). In all cases, a reduction
in complex I activity resulted in significantly increased Tim23-2
protein abundance. Collectively, these results show that the
amounts of complex I and Tim23-2 protein abundance appear to
be linked in Arabidopsis. Although the link between the amount
of complex I and Tim17:23 was unexpected, it is likely that this
occurs given that these two complexes share two proteins.
Therefore, a mutation that affects complex I assembly, but not
the amount of B14.7 directly, may result in additional B14.7
protein being available for assembly into TIM17:23, to replace
Tim23-2. In this case, it is proposed that the other subunits of
the TIM17:23 complex, which were observed to increase in
abundance, compensate to assemble all of the available Tim23-2.
However, in tim23-2 OE plants, it appears that this leads to instability of complex I. The mechanism by which this occurs is
unclear but may be explained by the fact that B14.7 is predicted
to be on the periphery of complex I (Klodmann et al., 2010;
Klodmann and Braun, 2011). Thus, an interaction between
complex I and Tim17:23, and Tim17:23 and complex III may
facilitate the formation of a supercomplex of complex I+III, which
is routinely observed in plant mitochondria (Meyer et al., 2008;
Klodmann et al., 2011). However, in the presence of excess
Tim23-2, the dynamics of this interaction or equilibrium are altered, leading to instability of complex I (Figure 4). Note that in
the tim23 OE plants, a new complex (MIC) is observed, and this
complex is not an assembly intermediate or degradation product of complex I, as analysis of this complex reveals that it
contains a variety of proteins (see Supplemental Figure 4 online).
Figure 8. (continued).
(A) Venn diagrams showing the overlap of significantly changing genes at a fold change cutoff of 1.5-fold for rug mutants (Kühn et al., 2011) and tim23-2
OE lines. Red indicates induced transcripts and blue indicates downregulated transcripts.
(B) Overview of significant changes (FDR-corrected P < 0.05) in transcript abundance, in response to overexpression of Tim23-2, for transcripts
associated with mitochondrial function and organelle biogenesis. Red boxes indicate transcripts that are induced in tim23-2 OE lines, and blue boxes
show transcripts that are downregulated. Color scale is shown as log2 fold change.
(C) Quantitative RT-PCR was performed to compare the abundances of mitochondrial transcripts in tim23-2 OE and KO seedlings compared with Col-0
seedlings. Transcript levels are depicted as log2 of the ratio of values determined in mutant versus Col-0 samples for each transcript on the mitochondrial genome. Three technical replicates of three independent biological repeats were performed and averaged per genotype; standard errors are
indicated. Nuclear 18S (used as a normalization control) along with ACT2 and UBC transcripts were included as controls.
(D) In organellar translations in mitochondria isolated from Col-0, tim23-2 OE, and KO lines. Equal amounts of mitochondria were used in assays and
the incorporation of [35S]Met was monitored in proteins over time.
(E) The 76 genes defined as highly coexpressed with tim23-2 were categorized according to function to determine over- or underrepresented groups
compared with the whole genome. Categories shown in bold red and blue indicate significantly (P < 0.0001, x2 test) over- and underrepresented
categories, respectively, compared with the whole genome. Percentages shown in parentheses indicate the relevant percentage observed for a whole
genome categorization (see Supplemental Tables 1 and 2 online).
16 of 21
The Plant Cell
We propose that this MIC complex results from the increased
expression of a large variety of mitochondrial proteins that are
imported but not assembled into functional complexes. To our
knowledge, nonassembled proteins have not previously been
detected in chemical amounts in mitochondrial complexes that
contain several diverse proteins.
The increased abundance of Tim23-2 protein in the tim23-2
OE line was accompanied by additional changes that together
can be grouped into the process of mitochondrial biogenesis. It
was notable that transcripts encoding many of the components
of the mitochondrial respiratory chain were significantly upregulated in abundance in the tim23-2 OE line (Figure 8). An
analysis of a variety of transcriptome data sets also reveals that
this upregulation of respiratory chain components is quite
unique (Giraud et al., 2012), as generally transcripts encoding
respiratory chain components are stable under a variety of
stresses (Clifton et al., 2005) or even in KOs of complex I components (Meyer et al., 2009). Furthermore, there are two additional features of the tim23-2 OE plants that are worth noting:
First, a comparison of the protein changes observed with quantitative immunoblotting reveals that the increase in Tom20-2,
Tom20-3, and Tom20-4 at a protein level is not accompanied by
an increase in the transcript abundance of genes encoding
these proteins. This suggests that the protein abundance is
regulated at a posttranscriptional level. In a previous study,
when any one or two of the Tom20 isoforms in Arabidopsis were
inactivated, an increase in the other isoforms was observed,
again without any change in transcript abundance (Lister et al.,
2007), and consistent with posttranscriptional regulation of
protein abundance for these components. Similarly, the increase
of Tim23-2 protein abundance in the tim23 OE plants also appears to occur at a posttranscriptional level, supporting the
findings from several previous studies indicating that the
abundance of mitochondrial protein import components occurs
posttranscriptionally (Howell et al., 2006, 2007; Law et al., 2012).
The insertion of the T-DNA in the 59 untranslated region may
alter translation of Tim23-2 mRNA, leading to the increase in
protein abundance observed in this study. Studies in several
plants have revealed a role for the 59 untranslated region in
determining translation efficiency (Kawaguchi and Bailey-Serres,
2005; Wever et al., 2010).
Note that examining genes located in close proximity to
Tim23-2 from microarray data revealed that transcript abundance for these genes were largely unaffected. Second, while
there is a significant overlap in the transcriptome response between the tim23-2 OE line and the complex I mutant rug line,
there are also responses that are unique to both (Figure 8A). The
response to both mutations displays common features, such as
a large reduction in complex I activity, an increase in Tim23-2
abundance and the abundance of various other proteins of the
protein import apparatus, and many common changes in the
transcriptome. However, differences were also apparent, including the finding that mitochondria from the complex I rug
mutant also displays an increase in the rate of protein imported
via the carrier import pathway and transcripts encoding components of the mitochondrial respiratory chain are not upregulated in this mutant. Finally, the overexpression of yeast Tim23 in
yeast cells using the galactose promoter to drive expression
resulted in no detrimental effects (Emtage and Jensen, 1993;
Bauer et al., 1996), unlike what we have seen for plants in this
study. It is proposed that the difference between plants (and
likely other organisms, such as yeast) may be due to the fact
that mitochondrial biogenesis and respiratory chain activity in
yeast are not linked, given that yeast is a facultative anaerobic
organism. Thus, it is not surprising that the expression of the
respiratory chain components and mitochondrial biogenesis are
not coupled. Rather, in yeast, the expression of mitochondrial
respiratory chain components is under the control of hemeactivated proteins that are in turn regulated by the availability of
oxygen (Kwast et al., 1998). However, mitochondrial biogenesis
still occurs in the absence of oxygen; thus, the respiratory chain
and mitochondrial biogenesis are not coupled in yeast. By
contrast, respiratory chain activity and mitochondrial biogenesis
may be linked in multicellular organisms. Thus, the interaction
reported in this study, between complex I and TIM17:23 in
Arabidopsis, provides a mechanism to link mitochondrial biogenesis and activity.
In conclusion, the results presented here show that in Arabidopsis, the B14.7 and Tim23-2 proteins are imported into and
accumulate in two mitochondrial protein complexes: the respiratory
chain complex I and the protein import complex TIM17:23. It is also
seen that the abundance of complex I is inversely related to the
abundance of Tim23. This may provide for an elegant system to
regulate mitochondrial biogenesis, as any changes in complex I,
due to oxidative damage or increased respiratory demand, could
trigger signals that increase the amount of Tim23-2 protein and
thus activate mitochondrial biogenesis.
METHODS
Gene Identification and Phylogenetic Analysis of Proteins
The Mitochondrial Protein Import Components database (Lister et al.,
2003) and The Arabidopsis Information Resource (www.Arabidopsis.org)
was used to identify genes used in this study. Alignments were performed
using ClustalW2 (www.ebi.ac.uk).
Plant Material and Culture Conditions
The SALK T-DNA insertion lines were obtained from the ABRC seed stock
center (Alonso et al., 2003), and GABI lines were obtained from GABI-Kat
(Rosso et al., 2003) for Tim23-2 (SALK_143656 and GABI_689C11). Lines
were screened for homozygosity of the T-DNA insert with primers listed in
Supplemental Table 3 online, as previously outlined (Carrie et al., 2010b;
Kühn et al., 2011). T-DNA mutants and the wild type were germinated on
0.53 Gamborg’s B5 salt medium containing 3% (w/v) Suc grown at 22°C
with a light intensity of 80 mmol quanta m22 s21 in a 16-h photoperiod.
DNA and PCR
Genomic DNA was isolated from 14-d-old plants, and PCR was performed using the REDExtract-N-Amp Plant PCR kit (Sigma-Aldrich)
according to the manufacturer’s instructions. Cloning from cDNA was
performed as previously described (Murcha et al., 2003) using primers
listed in Supplemental Table 3 online.
Isolation of Mitochondria from Arabidopsis thaliana Plants
For the isolation of mitochondria from water-cultured plants, mutant and
Col-0 seeds were sterilized and inoculated in half-strength Murashige and
A Link between Mitochondrial Activity and Biogenesis
Skoog media, containing 0.53 Gamborg’s B5 salts, 3% (w/v) Suc, 50 µg/
mL cefotaxime, and 2 mM MES KOH, pH 5.7. Plants were grown for
2 weeks as described above and mitochondria isolated as described
previously (Lister et al., 2007).
BN-PAGE and Immunodetection of Protein Complexes
BN-PAGE was performed according to Jänsch et al. (1996) as described
by Meyer et al. (2009). Following separation, gels were stained with
Coomassie Brilliant Blue, subjected to an in-gel complex I activity assay,
or transferred to polyvinyl difluoride membrane and immunodetected with
a selection of antibodies as described below.
In-Gel Complex I Activity Assay
The assay was performed according to Zerbetto et al. (1997).
Immunoblot Analysis
Mitochondrial protein was separated by SDS-PAGE and transferred
to Hybond-C extra nitrocellulose membrane (GE Healthcare). Immunodetection of proteins was performed as described previously (Murcha
et al., 2005a). The linearity of the response of the antibodies were tested
with 10, 20, and 40 mg of mitochondrial proteins (see Supplemental Figure
5A online) and equal protein loading confirmed by staining with Coomassie Brilliant Blue (see Supplemental Figure 5B online). Furthermore,
the CV was calculated for fold changes in abundances of porin and Tom40
to ensure that they were of equal abundance in samples. The CV was
determined by calculating the ratio of the standard deviation of the mean,
providing a statistical indication of the spread/variation in abundance.
Antibodies used were raised against Tim17-2 (Murcha et al., 2005a),
Tom20-2, 20-3, 20-4, Metaxin (Lister et al., 2007), Tom40, RISP, Sam50
(Carrie et al., 2010b), AOX, a-subunit of ATP synthase, Porin (Elthon et al.,
1989), Nad9 (Lamattina et al., 1993), COXII, cytochrome c, b-subunit of
ATP synthase (Agrisera), and Ndufs4 (Meyer et al., 2009). For the remaining antibodies, recombinant proteins of Tim9 (At3g46560, amino
acids 1 to 93), Tim50 (At1g55900, amino acids 110 to 269), Tim44
(At2g36070, amino acids 100 to 472), Tim21 (At4g00026, amino acids 160
to 376), Tim23-2 (At1g72750, amino acids 3 to 32 and 103 to 143), Tim23-3
(At3g04800, amino acids 6 to 32 and 64 to 132), At3g25120 (amino
acids, amino acids 89 to 189), were expressed and purified as previously
outlined (Carrie et al., 2010b). For antibodies raised against B14.7
(At2g42210) (amino acids 1 to 100) and Tim22 (At3g10110/At1g18320
amino acids 1 to 50), cDNA was cloned into pDest15 using Gateway
technology, and glutathione S-transferase–tagged recombinant proteins
were expressed, bound to GST-Sepharose (Scientifix), and electroeluted.
Recombinant proteins were confirmed by mass spectrometry prior to
inoculation into rabbits as per the standard protocol (Cooper and Paterson,
2008). Immunodetection of newly synthesized antibodies was performed
at a 1 in 5000 dilution against purified mitochondria.
In Vitro Import Studies
Mitochondria for in vitro import experiments were prepared from 14-d-old
Arabidopsis seedlings grown in liquid culture, as previously described
(Lister et al., 2007). [35S]Met-labeled precursor proteins of AOX (X68702)
(Whelan et al., 1995), FAd subunit of ATP synthase (X74296) (Smith et al.,
1994), ANT (X57556), Pic (AB016063) (Murcha et al., 2005a), and MDHAR
(At3g09940) (Chew et al., 2003) synthesized using the rabbit reticulocyte
TNT in vitro transcription/translation lysate (Promega). Import of radiolabeled precursor protein into isolated mitochondria was performed as
previously described (Lister et al., 2007) at increasing time points. Following import, mitochondria were pelleted at 20,000g for 5 min and
subjected to SDS-PAGE analysis. Gels were stained in Coomassie
17 of 21
Brilliant Blue, dried, and exposed to a BAS TR2040 phosphor-imaging
plate (Fuji) for 24 h. The exposed plate was visualized using the BAS 2500
Bio-Imaging Analyzer (Fuji). Proteinase K–protected, mature, radiolabeled
imported proteins were quantified at each time point and normalized to
the highest time point for the wild type, which was set to 1 using Quantity
One software (Bio-Rad) (Lister et al., 2007). Three biological replicates
were performed for each mutant and precursor protein and the average
(and SE) of import amount relative to Col-0 was determined.
Imports analyzed by BN-PAGE were performed as previously described (Carrie et al., 2010b). [35S]Met-labeled precursor proteins of
Tim17-2 (At2g37410), Tim23-2 (At1g72750) (Murcha et al., 2005a), Tim21
(At4g00026), complex I subunit At1g4726, MPPa (At1g51980) (Kühn et al.,
2011), B14.7 complex I subunit At2g42210 (Murcha et al., 2007), and
cytochrome c1 subunit of ubiquinone cytochrome c reductase (At4g32470)
were generated from Arabidopsis cDNA cloned into pDest14 using Gateway technology (Invitrogen) and used in import assays. Precursor proteins
were synthesized and imported as described previously except that 250 mg
of mitochondrial protein was used per time point. Following import, mitochondria were pelleted at 20,000g for 5 min and subjected to BN-PAGE
analysis as detailed above. BN-PAGE gels were fixed in 40% (v/v) methanol
and 10% (v/v) acetic acid and visualized by autoradiography, as described
above.
RNA Isolation and Global Transcript Analysis
Analysis of the changes in transcript abundance between Col-0, tim23-2
OE, and tim23-2 KO lines was performed using Affymetrix GeneChip
Arabidopsis ATH1 Genome Arrays (Affymetrix). Whole rosettes from
several seedlings at the 8 true leaf stage of development, grown under
a 16-h photoperiod, were pooled for each biological replicate. Col-0 and
mutant tissue samples were collected in biological triplicate. For each
replicate, total RNA was isolated and microarrays were performed as
described (Kühn et al., 2011).
Quantitative RT-PCR
RNA, isolated as described above, from Col-0, tim23-2 OE, and tim23-2
KO plants was subjected to two consecutive treatments with TURBO
DNase (Ambion), confirmed by PCR to be free of detectable amounts of
DNA, and reverse transcribed exactly as described (Kühn et al., 2009).
Quantitative RT-PCR on the mitochondrial transcriptome was performed
using the LightCycler 480 SYBR Green I Master Mix (Roche Applied
Science) using the PCR program and primer pairs and analyzed as described (Kühn et al., 2009).
Statistical Analysis of Microarray Data
Data quality was assessed using GCOS 1.4 before CEL files were
exported into AVADIS Prophetic (version 4.3; Strand Genomics) and
Partek Genomics Suite software, version 6.3, for further analysis. CEL files
are available at Array Express (http://www.ebi.ac.uk/arrayexpress/) under
the accession number E-MEXP-3309. Public array data for the rug mutant
(Kühn et al., 2011) was also independently analyzed and included. MAS5
normalization algorithms were performed only to generate present/absent
calls across the arrays. Probe sets that recorded absent calls in two or
more of the three biological replicates for all four genotypes conditions
were removed from further analysis. Bacterial controls, probe sets for
organelle transcripts and ambiguous probe sets for multiple transcripts
were also removed, resulting in a final set of 14,831 probe identifiers for
downstream analysis. CEL files were also subjected to GC content
background robust multiarray average normalization for computing
fluorescence intensity values used in differential expression analyses.
Correlation plots were examined between all arrays using the scatterplot
18 of 21
The Plant Cell
function in the Partek Genomics Suite, and in all cases r > 0.98 (data not
shown). Analysis of differential expression was performed using a regularized t test based on a Bayesian statistical framework using the software
program Cyber-T (Baldi and Long, 2001) (http://cybert.microarray.ics.uci.
edu/). Cyber-T employs a mixture model-based method described by
Allison et al. (2006) for the computation of the global false-positive and
false-negative levels inherent in a DNA microarray experiment. To control
for FDR accurately and minimize false positives within the differential
expression analysis, PPDE(P) values and PPDE(>P) values were calculated, as a means to measure the true discovery rate (1 – FDR). Changes in
transcript abundance were considered significant with a PPDE(>P) > 0.95
and a fold change >1.5-fold. Overlaps in the transcript abundance responses for the different genotypes were plotted on Venn diagrams to
determine statistically significant over- or underrepresentation in the
overlap, compared with that which is expected by random chance, using
a Pearson’s x2 test for independence. Transcript abundance changes for
mitochondrial proteins were visualized in a custom MapMan pathway
(Thimm et al., 2004). A nonredundant list of 76 transcripts that are the
most highly coexpressed transcripts, genome wide, against Tim23-2 was
created from the top 50 gene hits from BAR Expression Angler and The
Arabidopsis Coexpression Data Mining Tools (coexpression multiexperiment tool, http://www.Arabidopsis.leeds.ac.uk/act/coexpanalyser.
php#CO2) shown in Supplemental Table 2 online (Manfield et al., 2006).
Cocorrelation scatterplots were generated using the cocorrelation 2-D
Pearson correlation coefficients plot, with the 76 coexpressed top hit
transcripts highlighted. Functional categorization analysis was performed
using simplified annotation categories defined in MapMan software bins to
define over/underrepresented functional groups based on percentile
distributions of the whole genome.
In Organello Protein Synthesis Assays
Mitochondria were isolated as described above and protein synthesis
reactions were performed as previously described (Giegé et al., 2005).
Blue Native Elution and in-Gel Digestion
The complex of interest was excised from Blue native gels and proteins
extracted by electroelution. Gel slices were minced by passing back and
forth between two syringes and added to the H-shaped eluter vessel of
a CBS Scientific electroeluter. Proteins were eluted according to the
technique detailed previously (Hunkapiller et al., 1983) Protein identification was performed by in-gel digestion and electrospray ionization
tandem mass spectrometry according to previous methods (Meyer et al.,
2007).
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: Tim17-1 (At1g20350), Tim17-2 (At2g37410), Tim17-3 (At5g11690),
Tim23-1 (At1g17530), Tim23-2 (At1g72750), Tim23-3 (At3g04800), B14.7
(At2g42210), MPPa (At3g16480), Tim50 (At1g55900), Ndufs4 (At5g67590),
RISP (At5g13430), COXII (AtMg00160), a-ATP synthase (At2g07698), rug
(At5g60870), Rpotmp (At5g15700), Tim22 (At1g18320/At3g10110), Tim44
(At2g36070), Tom20-2 (At1g27390), Tom20-3 (At3g27080), Tom20-4
(At5g40930), Sam50 (At5g05520), Tim21 (At4g00026), Tim9 (At3g46560),
Tom40 (At3g20000), cyc1-1 (At3g27240), and cyc1-2 (At5g40810).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Determining the Specificity of B14.7 Antibody.
Supplemental Figure 2. Interaction of Tim21 with Complex III in
Arabidopsis.
Supplemental Figure 3. Analysis of Tim23 Protein and Transcript
Abundance in T-DNA Insertion Lines.
Supplemental Figure 4. Identification of the Mixed Inner Membrane
Complex in the tim23-2 OE Line.
Yeast Two-Hybrid Protein Interactions
Yeast two-hybrid interaction assays were performed as described (Giraud
et al., 2010). AH109 cells transformed with pGAD Tim23-2 were mated
with Y187 cells transformed with pGBK Tim17-2, pGBK Tim23-2, pGBK
Tim50, pGBK B14.7, pGBK Tim21, pGBK MPPa, and empty pGBK. Serial
dilutions of yeast growing on auxothrophic selection media (2Leu –Trp
–His –Ade) were regrown on selection plates for diploid selection (–Trp
–Leu) and positive interaction selection (–Trp –Leu -Ade –His and –Leu
–Trp –Ade –His with X-a-D-galactoside).
Supplemental Figure 5. Determining the Linearity of Antibodies Used
and Protein Abundance in Mitochondrial Isolations.
Supplemental Table 1. Summary of Changes in Transcript Abundance for Nuclear Genes Encoding Mitochondrial Import and Assembly Components.
Supplemental Table 2. Summary of 76 Transcripts Defined as Being
Highly Coexpressed with tim23-2 across Public Arabidopsis Microarray Data Sets.
Supplemental Table 3. List of Primers Used in This Study.
Immunoprecipitations
Two hundred micrograms of mitochondria was solubilized in digitonin
buffer consisting of 30 mM HEPES KOH, pH 7.4, 150 mM potassium
acetate, 10% (v/v) glycerol, and 1 mg digitonin and placed on ice for
20 min. One hundred microliters of Protein A Sepharose with or without
10 mL of antibody was added with solubilization buffer without digitonin,
including 0.5% (w/v) BSA and Complete Protease inhibitor (Roche), and
incubated for 3 h at 4°C. Beads were washed and samples resolved
by SDS-PAGE, transferred to nitrocellulose membrane, and immunodetected with antibodies indicated.
ACKNOWLEDGMENTS
This work was supported by the University of Western Australia
Scholarship for International Research Fees grant to Y.W., an Australian
Research Council Australian Postdoctoral Fellowship grant to M.W.M.,
and the Australian Research Council Centre of Excellence Program
CEO561495.
AUTHOR CONTRIBUTIONS
BiFC
BiFC was performed as outlined previously (Citovsky et al., 2008) in onion
(Allium cepa) epidermal cells. Images were captured using Cell imaging
software (Olympus) as previously described (Carrie et al., 2009).
J.W. and M.W..M. designed the experiments and wrote the article. Y.W.,
C.C., D.E., O.D., and M.W.M. performed the experimental procedures
and carried out the data analysis. E.G. and R.N. carried out gene
expression and transcriptome analysis.
A Link between Mitochondrial Activity and Biogenesis
Received March 27, 2012; revised May 22, 2012; accepted June 11,
2012; published June 22, 2012.
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Dual Location of the Mitochondrial Preprotein Transporters B14.7 and Tim23-2 in Complex I and
the TIM17:23 Complex in Arabidopsis Links Mitochondrial Activity and Biogenesis
Yan Wang, Chris Carrie, Estelle Giraud, Dina Elhafez, Reena Narsai, Owen Duncan, James Whelan and
Monika W. Murcha
Plant Cell; originally published online June 22, 2012;
DOI 10.1105/tpc.112.098731
This information is current as of June 15, 2017
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